Dendritic cells, potent antigen presenting cells, play an essential role in initiating T-cell responses. Using a battery of in vitro assays and flow cytometry for guidance, we have markedly modified the protocol used at the NIH to prepare dendritic cells for clinical immunotherapy trials. In particular, we have shortened the duration of incubation of DCs with GM-CSF/IL4 in vitro from 7 to 3 days and used a different combination of (LPS and IFN-gamma; instead of CD40L and and IFN-gamma) to promote dendritic cell maturation. Based on comparisons of DC maturation marker expression, cytokine production, and T cell stimulating activity, we can clearly show these changes significantly improve dendritic cell phenotype and function. Consequently, this methodology has been adopted as the standard for DC preparation within the Department of Transfusion Medicine at the NIH.[unreadable] [unreadable] During the past year, Institute investigators have initiated two new clinical protocols using a modified DC preparation method to prepare clinical products. One of these uses dendritic cells pulsed with immunogenic peptides to immunize patients with refractory ALL against the tumor related antigen WT1. The second protocol uses dendritic cells pulsed with a lysate derived from the patients own tumor to immunize patients against recurrent sarcomas. Both protocols are in their early stages hence no information about clinical or in vitro responses is as yet available.